Schistosomiasis is a vascular parasitic disease in humans caused by blood flukes of the schistosoma species. The disease afflicts an estimated 200 million people worldwide with approximately 500-600 million people at risk of infection. Schistosomiasis is one of many helminthic diseases infecting over a billion people worldwide. These diseases include ascariasis, trichuriasis, enterobiasis, filariasis, trichinosis, onchocerciasis, fascioliasis, and cysticercosis. Schistosomiasis ranks second to malaria as a major cause of morbidity and suffering due to parasites.
The means of detecting parasitic helminthic infections in general is highly variable. Some methods rely on direct detection of eggs or parasitic debris in feces and urine. This is exemplified by diagnostic procedures for Taenia saginata, Hymenolepsis nana, Diphyllobothrium latum, Schistosoma species, Fasciola hepatica, Trichuris trichiura, Gastrodiscoides hominis, Ascaris lumbricoides, common hookworms (Necator americanus, Ancylostoma duodenale, and A. ceylanicum). In some diseases diagnosis is based on examination of blood or skin scrapings and direct microscopic identification of parasites, such as for the filarial parasites, Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus. Immunological and serological tests are sometimes also used for certain parasitic helminths, and they can be effective in aiding the diagnosis. Serological tests are used, for example, in diagnosis of Visceral Larva Migrans or VLM (a syndrome caused by migrating larvae of Toxocara canis and T. cati), Trichinella spiralis, and some filarial parasites mentioned above.
Thus, specific immunological assays for diagnosis and treatment are not common and when available, the specific antigens used for immunological detection are not chemically defined. In addition, the serological tests may provide falsely positive information about the presence of active infections, since prior infection may result in positive serological tests, due to prior immunization by parasite-derived antigens.
There is no vaccine to prevent schistosomiasis (or any other major human parasitic infection), although patients suffering from schistosomiasis can be effectively treated in most cases with certain schistosomicides to eliminate the existing infection. However, re-infection in endemic areas is common, and there is evidence for the development of drug-resistant schistosome strains. It is hoped that the availability of the modern tools of biochemistry, immunology, and cell and molecular biology will foster new developments that are needed to facilitate the production of new diagnostic assays, effective vaccines and preventatives, interventional strategies and new therapeutics. In addition, basic information learned about these parasites may have added benefits and may shed light on fundamental molecular mechanisms regulating human immunity and inflammation.
Background on Schistosome Biology
Schistosomes are dioecious and digenetic trematodes of the family Schistosomatidae and over 85 species of schistosomes exist. The life cycle alternates between a definitive vertebrate host and an intermediate freshwater snail host. Mammals are infected by schistosome of the genus Schistosoma, whereas birds are infected by several related genera (e.g., Ornithobilharzia and Austrogbilharzia). (The so-called "swimmers itch" in people is caused by transient skin infection with bird schisotosomes, although no permanent infection is generated.) Three of the major species that infect humans are S. mansoni, S. japonicum and S. haematobium. Adult schistosomes (.about.0.5-1.5 cm in length) dwell as pairs of males and females in veins of their definitive host and each species localizes to a specific organ--S. mansoni in the portal veins draining the large intestine, S. japonicum in the veins of the small intestine and S. haematobium in the urinary bladder plexus. Adult worms can live for many years and the females are prodigious egg layers. It is estimated that every 10 minutes S. haematobium, S. mansoni and S. japonicum release approximately 1, 2, and 10 eggs, respectively. The elliptically-shaped eggs range in size from 114 to 175 .mu.m by 40 to 70 .mu.m and have spines distinctive of each species. The worms have elongated bodies with an oral and a ventral sucker and a rough tegumental surface with numerous spiny protrusions.
The major pathology identified with schistosomiasis is associated with immune responses to the eggs. The eggs released by adult worms have several possible fates. Eggs that adhere to venule walls may cause a small blood thrombus (clot) that is slowly infiltrated by fibroblasts and overgrown by endothelial cells, which may be stimulated to proliferate. Non-adherent eggs may be swept back to the liver where they lodge in the hepatic capillary bed where they can cause hepatosplenomegaly and portal hypertension. If the adherent eggs successfully penetrate the vessels that lie close to the gut or bladder they may enter the feces or urine. About 2/3+L of the eggs fail to penetrate completely and become lodged in the gut and bladder walls. Eggs trapped within the liver and other tissues induce granulomatous response that are largely cell-mediated immune responses to secreted egg antigens, including glycoproteins. Egg secretions produced partly by the developing miracidia exit through micropores in the eggshell and proteins, glycoproteins, and polysaccharides in these secretions elicit immune/inflammatory responses in the definitive host.
Those eggs exiting in the urine and feces of the vertebrate definitive host hatch in freshwater into miracidia that infect specific snail intermediate hosts to continue the life cycle. In the snails the replicative stage occurs asexually and eventually free-swimming cercariae are released into the surrounding water upon appropriate stimuli, such as light. Cercariae have a bifurcated tail (.about.200 um) and a body (.about.125 um) containing oral and ventral suckers important for attachment to the skin of definitive hosts. Free-swimming cercariae seek the skins of warm-blooded animals and bind the skin and attach through their oral sucker. The cercariae then contract and copious amounts of mucus are secreted from the postacetabular glands onto the skin surface. The cercariae penetrate the skin and lose their tail through violent shaking and then undergo a phenomenal transformation into a schistosomula, in which there is a rapid change from aerobic to anaerobic metabolism. The transformation is also accompanied by pronounced changes in shape of the parasite and expression of surface glycoconjugates, and loss or exposure of many surface glycoproteins within hours. The schistosomula eventually leave the skin, some enter the peripheral circulation and are swept to the heart, whereas others may enter the lymphatic system and exit via the thoracic duct. The schistosomula have an arduous and complex migration with the vertebrate hosts over a period of many days, moving through the lung and liver, until they finally come to reside in the systemic circulation. There the worms sexually mature, male and female pairing occurs and egg laying commences within 4-5 weeks.
Pathology Associated with Schistosomiasis
Schistosomiasis can cause profound pathology in infected humans and animals. The acute phase, coinciding with the onset of egg-laying, is characterized by fever, dysentery, allergic reactions with occasional abdominal pain and liver tenderness, and severe eosinophilia, which may be followed by leukopenia. Acute schistosomiasis caused by S. japonicum is often accompanied by severe symptoms including fevers and chills (Katayama fever) Chronic schistosomiasis arises slowly and is accompanied by pathological changes in affected organs. Organ damage can result from the T cell-mediated granulomatous response to the eggs. In S. mansoni and S. japonicum infections there is often portal hypertension and gross enlargement of the liver and spleen and in some cases there may be pipestem fibrosis of the liver. In S. haematobium infections there is often terminal hematuria and sometimes dysuria. Also, in S. haematobium bladder cancer may occur as a secondary consequence.
Diagnosis of Schistosomiasis
The most common way to diagnose schistosomiasis is identifying eggs in urine and stool samples. The numbers of eggs and the egg morphology can be used to estimate approximate numbers of egg-laying worm pairs in the individual and to identify the species of schistosome causing the infection, respectively. This assay, however, has many drawbacks. It is laborious, requires collection and handling of stool samples, and requires skilled personnel using microscopic examination. In addition, stool samples from individuals with light to moderate infections may appear negative. A second method to diagnose schistosomiasis is the presence of hematuria in urine samples, caused by S. haematobium. Of course, this latter diagnostic assay is specific only for this species. Some serologic and immunologic assays have been developed, generally based on egg-derived antigens, but these have generally failed to demonstrate the necessary specificity and sensitivity. There is some promise shown in immunologic assays based on detecting circulating glycoproteins in the blood of schistosome-infected animals (van Lieshout et al, 1995; 1997). However, in some published studies these assays have demonstrated specificity and sensitivity ranging around 50.degree. in both parameters (van Lieshout et al., 1995); in some studies sensitivity was only 20% (de Jonge et al, 1990). Unfortunately, none of these immunologic assays are commercially available and it is not possible to compare one assay to another. Clearly, it is desirable to develop more specific and sensitive assays that can also discriminate between different helminthic infections.
Treatments of Schistosomiasis
Most cases of schistosomiasis can be effectively cured by single dose treatment with the common platyhelminthicide praziquantel (Biltricide). Metrifonate (Bilarcil) may be more effective for specifically treating S. haematobium infections. The mechanisms of killing by these drugs are not well understood. Praziquantel causes increased expression of surface antigens , along with other biochemical effects, but the drug appears to be effective in killing parasites only when there is associated humoral immunity. Some of the major targets for praziquantel killing in association with humoral immunity are surface glycoproteins.
Expression of Le.sup.x and LDN--Related Glycans by Schistosomes and Host Immunity to the Parasite
It has long been appreciated that glycoconjugates derived from schistosome cercariae, adult worms and eggs are major antigens in infected hosts. Among the first parasite glycoconjugates identified as immunogenic were the circulating glycoproteins derived from gut of the schistosome (Nash, 1978; Nash et al., 1978; Deelder et al., 1980; Carlier et al., 1980). These glycoconjugates are designated the circulating anodic antigen (CAA) and the circulating cathodic antigen (CCA), the latter of which was originally identified as a proteoglycan-like molecule (Nash et al., 1977; Nash and Deelder, 1985).
Despite the acknowledged importance of schistosome glycoconjugates to the host immune response, it has only been in the past few years that detailed structures of schistosomal glycoconjugates have been defined, and our laboratory has been instrumental in developing this area. Many of the complex-type N-glycans in schistosome adult surface glycoproteins contain the Lewis x (Le.sup.x) and polyLe.sup.x antigens (Srivatsan et al., 1992a) and Lex antigen is also present in the secreted circulating cathodic antigen (CCA) (van Dam et al., 1994). These Le.sup.x -containing glycans are particularly important, since we found that sera from infected animals contains a high level of antibody of both IgG and IgM to the Le.sup.x antigen (Nyame et al., 1996, 1997).
Some of the schistosome-derived N-glycans contain the lacdiNAc (LDN) sequence GalNac.beta.1.fwdarw.4GlcNAc-R (Srivatsan et al., 1992b), which contrasts with the typical Gal-GlcNAc or LacNAc sequence (Neeleman et al., 1994). LDN and LDNF (GalNAc.beta.1.fwdarw.4[Fuc.alpha.1.fwdarw.3]GlcNAc-R) have been found in glycoproteins synthesized by dog heart worm Dirofilaria immitis (Kang et al., 1993) and in the intestinal nematode Haemonchus contortus (DeBose-Boyd et al., 1998). (Gal=galactose; Fuc=fucose; GlcNAc=N-acetylglucosamine; GalNAc=N-acetylgalactosamine).
A test for diagnosing parasitic helminth infections which is highly specific, especially for schistosoma species would be highly desirable.